In recent years, health care has improved dramatically, in large measures due to the availability and improvement in assays. Assays detect particular chemical constituents, i.e., ligands, which have been correlated or are associated with various disease conditions. Ligands are detected primarily through a binding reaction with a ligand binding specific substance which preferentially binds to the ligand and not to other chemical constituents which may be present in the sample to be tested. Through a variety of imaginative techniques, the presence or absence of such a binding reaction can be detected, and thus in turn the presence or absence of the ligand in the sample determined.
There are two major types of assays to which this claimed invention relates and these include immunoassays and hybridization assays. Immunoassays have been in existence longer and are based upon the specificity of the reaction between an antibody and an antigen for which the antibody is specific. Antibodies initially used where of polyclonal origin, e.g., they were produced in animals following a challenge by the antigen for which the antibodies were desired. Of late, following the innovative developments by Koller and Millstein in 1975 (Nature 225:1061), monoclonal antibodies have been preferred since they can be more easily produced and allow exquisite selection by affinity and avidity.
There further exists a variety of techniques for labeling the antibodies and/or antigens including the employment of isotopic labels, fluorescent molecules, chemiluminescent molecules, enzymes, light scattering particles, energy transfer, schemes between pairs of spectrally matched molecules, and the like. These techniques are well known in the art and need not be reviewed in detail here. It is worthy, however, to distinguish between two diverse types of assays; homogenous and heterogeneous assays. Homogeneous are most desired from an operational standpoint since the entire reaction, and an addition of reagents for the performance of the assay, take place in a single solution along with the final detection step. Accordingly, mechanical manipulations which are time consuming and can cause errors are avoided, however, the technical aspects of developing such an assay with the desired sensitivity are substantial. In contrast, numerous assays are performed on a heterogeneous basis in that certain steps are performed in one solution which generally includes some type of solid phase material. The reaction to be detected takes place either in solution or on the solid phase and is then followed by a separation step whereby unreacted components, and thus contaminating influences, may be effectively removed. The result is generally a higher level sensitivity at the expense of additional mechanical manipulations. Conventional heterogeneous assays have employed dipsticks which may be easily removed from the solution by hand, or large beads which similarly allow facile transfer. Smaller beads, generally of latex or similar materials, have been employed and have relied upon filter and/or centrifugal methods for their sequestration from the fluids. The concept of employing large magnetic particles has also been explored and is described by Smith et al. in U.S. Pat. Nos. 4,272,510 and 4,292,920. Specifically, Smith et al. describe the use of BB type particles and their removal from solution by the employment of electromagnetic energized nails for removing the solid phase from container to container. While somewhat inelegant, the Smith et al. method does have an advantage in that the container walls which often provide a contaminating influence are eliminated assuming, of course, the detectable reaction has taken place on the solid phase. The methods, however, suffer from substantial risk of loss of accuracy due to the failure to remove and/or transfer all solid phase particles to another container, particularly as would be the case with micromagnetic or ferrous particles. Such microparticles would be greatly preferred over the large beads described by Smith et al. because the present has a far greater surface area upon which reactions may take place. Sensitivity is accordingly dramatically improved.
Corning et al. has made available commercially a magnetic separation device which is intended for use with large, e.g., 12 mm by 75 mm, test tubes containing the assay reagent mixtures and the magnetic particle solid phase component. The Corning device has horizontal molded ridges for receiving the test tube whereby the particles become attracted to a side of the test tube allowing removal of the liquids. The design of the Corning device is not, however, optimized for use with small sample volumes and cannot be made optimal for such application therefore limiting its utility.
It is an object of the present invention to provide devices for use with assays employing magnetic microparticles which are capable of use with small volume assays or &gt;60 at a time.
It is another object of the present invention to provide devices which may be employed with microtiter type trays whereby a plurality of assays may be performed simultaneously.
It is yet another object of the present invention to provide an autoclavable separation devices for use with ferrous solid phase materials.
It is a still further object of the present invention to provide a magnetic separation device which is readily adaptable to automated pipetting systems.
It is still yet a further object of the present invention to provide a magnetic separation device which may be adapted to provide one, two or four ferrous particle attraction positions within each sample container.
Another class of assays of more recent vintage are those relying upon the hybridization of nucleic acid probes with target nucleic acids. The target nucleic acid is generally that associated with an infective organism, e.g., bacteria virus and like, although the detection of specific cellular genomes is also contemplated. By greatly simplified explanation, hybridization assays rely upon the greatly preferred pairings between complementary nucleotide bases. Specifically, the preferred pairings are between adenine and thymidine, guanine and cytidine. Each strand of deoxyribonucleic acid (DNA) is comprised of a series of the foregoing bases, while its complementary strand comprises a matching but complementary series of DNA bases. Thus, one can disassociate the double-stranded nucleic acid into single-strands and with probes comprised of nucleic acids having complementary sequences, one may produce double-stranded nucleic acid wherein the probe is hybridized to the target nucleic acid only at complementary positions.
DNA is transcribed into ribonucleic acid (RNA) which is also comprised of ribonucleotides of the same four bases with the exception that uracil is substituted for thymidine. Because of the manner of transcription, the RNA sequence is also complementary to the DNA sequence and accordingly comprises the same basic identifying information. Thus, one may similarly detect RNA of a microorganism or cell by hybridizing the target RNA to a probe comprising complementary sequence. The production and formulation of nucleic acid probes while a comparatively recent development, are still arts well known and well described in the literature. A helpful reference in this regard is Maniatis et al., a cloning manual, the relevant portions of which along with references referred to therein are incorporated herein by reference.
As may be readily appreciated, many of the same techniques employed with immunoassays are the labeling, and heterogeneous/homogenous schemes are applicable to hybridization assays. In particular, the employment of solid phase materials in heterogeneous assays are techniques which make hybridization assays especially useful. The utilization of micromagnetic particles is not, however, commonplace primarily in large measure due to the inapplicability of the present magnetic separation devices to hybridization assays.
It is, therefore, another object of the present invention to provide suitable magnetic separation devices useful with hybridization assays and ferrous solid phase particles.